Whereas conventional virus diagnosis has been based predominantly on the detection of viral antigens or specific antibodies thereto, in recent years attention has shifted towards methods for the direct detection of the genome of viruses or nucleic acid sequences derived thereof, both RNA and DNA. These methods are usually based on nucleic acid hybridization. Nucleic acid hybridization is based on the ability of two strands of nucleic acid containing complementary sequences to anneal to each other under the appropriate conditions, thus forming a double stranded structure. When the complementary strand is labeled, the label can be detected and is indicative for the presence of the target sequence. Especially in combination with methods for the amplification of nucleic acid sequences these methods have become an important tool in viral diagnosis, in particular for the detection of human immunodeficiency virus (HIV).
Nucleic acid amplification techniques are especially useful as an additional technique in cases where serological methods give doubtful results or in cases where there may be a considerable time period between infection and the development of antibodies to the virus. With HIV, seroconversion usually can occur some 3-6 months after exposure to the virus.
Thus, whereas no antibodies will be detected with conventional immunoassays, proviral DNA or circulating viral RNA may already be detectable. Also in monitoring antiviral therapy, methods based on nucleic acid amplification have several advantages over serological methods. Especially quantitative amplification methods provide a powerful tool in assessing the changes in the amount of virus present before and during therapy.
The choice of the oligonucleotides to be used as primers and probes in the amplification and detection of nucleic acid sequences is critical for the sensitivity and specificity of the assay.
The sequence to be amplified is usually only present in a sample (for example a blood sample obtained from a patient suspected of having a viral infection) in minute amounts. The primers should be sufficiently complementary to the target sequence to allow efficient amplification of the viral nucleic acid present in the sample. If the primers do not anneal properly (due to mispairing of the bases on the nucleotides in both strands) to the target sequence, amplification is seriously hampered. This will effect the sensitivity of the assay and may result in false negative test results. Due to the heterogeneity of viral genomes false negative test results may be obtained if the primers and probes are capable of recognizing sequences present in only part of the variants of the virus. The HIV virus shows a high heterogeneity. Genetic variability has been demonstrated amongst isolates from different continents but also between individuals and between different stages of the disease. Based on sequence analysis two groups within HIV-1 have been identified: group M (M for “major”), and group O (O for “outlier”). Within group M subtypes (A-H), each constituting a philogenetic separate set of sequences, have been assigned and additional ones are being identified. This sequence variation is not uniformly distributed throughout the genome. The HIV-1 genome, like all retroviral genomes, roughly consists of the following regions: The gag gene of the HIV-1 genome is the region encoding the core proteins of the virus (for example, p24). The env gene encodes a large precursor protein, gp160, which is processed into the envelop proteins gp120 and gp41. The pol gene encodes the polymerase of the virus (reverse transcriptase). The Long Terminal Repeat region's (LTR's) are the regions on the viral genome that participate in the integration of the virus with the host cell and in the regulation of transcription of the viral genes. Some regions are more prone to sequence variation than others. Especially in the env domain sequence variation can be as high as 30% between members of the different subtypes. Ideally, primer selection should be based on knowledge of interstrain variability in candidate primer sequences and the consequences of mismatching at primer sites. McCutchan et al, J. AIDS, 4, 1241-1250, 1991, used PCR to make a genetic comparison of different HIV-1 isolates. Using anchored PCR (varying sense primers were used with a constant antisense primer. primers were chosen from relatively conserved regions in gag, env and LTR) The effect of primer mispairing on the amount of PCR product obtained was also investigated. Mispairing at the 3′ end of the primer decreased the amount of product sometimes more then 100-fold.
The detection of all presently known subtypes of HIV-1 is of extreme importance, especially with regard to patient management, security of blood and blood products and clinical and epidemiological studies. Current assays for the amplification and subsequent detection of HIV-1 derived nucleic acid sequences are usually based on amplification of sequences in the gag region of the viral genome. These assays have been developed for subtype B, which is the major subtype in European countries and the United States. However, the presence of other subtypes, which were geographically confined before, is increasing due to frequent travel between these countries and, for example, African countries. Sensitive assays are therefore needed that are capable of detecting as much variants of the HIV-1 virus as possible (preferably all).
Research aimed at identifying suitable primer sets for the reliable amplification of HIV-1 derived nucleic acid sequences has been ongoing for the past years. Engelbrecht et al., J. Virol. Meth., 55, 391-400, 1995, describe a study aimed at the development of a specific and sensitive PCR protocol using env, gag and LTR primer pairs to detect subtypes present in the Western Cape, South Africa. Twenty four strains of which it was known that they belonged to subtypes B, C and D were analyzed. It was found that the performance of the primer pairs was greatly dependent on the optimization of the reaction conditions for the different primer pairs. Only when less stringent conditions were used (for example, with the LTR primer pair an increased cycle time and lower annealing temperatures were required) these particular strains of HIV could be detected with sufficient sensitivity and reproducibility with all primer pairs.
Zazzi et al., J. Med. Virol., 38, 172-174, 1992, developed a two-step PCR reaction (using nested primers) for the detection of HIV-1 DNA in clinical samples. The primers used for amplification were derived from the gag gene and the LTR region. The patients tested in this study were all from neighbouring areas, which makes it likely that they represent only a limited number of different viral strains.
A quantitative PCR method using LTR derived nested primers was described by Vener et al. in BioTechniques, 21, 248-255, 1996. This procedure was only tested on HIV-1MN infected peripheral blood mononuclear cells (PBMC). Thus nothing can be said about the suitability of the primers used for detecting different subtypes of the virus.